Chemokines (chemoattractant cytokines) comprise a family of structurally related secreted proteins of about 70-110 amino acids that share the ability to induce migration and activation of specific types of blood cells (reviewed in Baggiolini M., et al. (1997) Annu. Rev. inmmunol. 15: 675-705; Proost P., et al. (1996) Int. J. Clin. Lab. Rse. 26: 211-223; Premack, et al. (1996) Nature Medicine 2: 1174-1178; Yoshie, et al. (1997) J. Leukocyte Biol. 62: 634-644). Over 30 different human chemokines have been described to date. They vary in their specificities for different leukocyte types (neutrophils, monocytes, eosinophils, basophils, lymphocytes, dendritic cells, etc.), and in the types of cells and tissues where the chemokines are synthesized. Chemokines are typically produced at sites of tissue injury or stress, where they promote the infiltration of leukocytes into tissues and facilitate an inflammatory response. Some chemokines act selectively on immune system cells such as subsets of T-cells or B lymphocytes or antigen presenting cells, and may thereby promote immune responses to antigens. Some chemokines also have the ability to regulate the growth or migration of hematopoietic progenitor and stem cells that normally differentiate into specific leukocyte types, thereby regulating leukocyte numbers in the blood.
The activities of chemokines are mediated by cell surface receptors which are members of the family of seven transmembrane, G-protein coupled receptors. At present, over twelve different human chemokine receptors are known, including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR1, CXCR2, CXCR3, and CXCR4. These receptors vary in their specificites for specific chemokines. Some receptors bind to a single known chemokine, while others bind to multiple chemokines. Binding of a chemokine to its receptor typically induces intracellular signaling responses such as a transient rise in cytosolic calcium concentration, followed by cellular biological responses such as chemotaxis. Some chemokine receptors also serve as coreceptors for HIV, such that they interact with HIV and with the cellular CD4 receptor to facilitate viral entry into cells.
Chemokines are important in medicine because they regulate the movement and biological activities of leukocytes in many disease situations, including, but not limited to: allergic disorders, autoimmune diseases, ischemia/reperfusiion injury, development of atherosclerotic plaques, cancer (including mobilization of hematopoietic stem cells for use in chemotherapy or myeloprotection during chemotherapy), chronic inflammatory disorders, chronic rejection of transplanted organs or tissue grafts, chronic myelogenous leukemia, and infection by HIV and other pathogens.
Antagonists of chemokine receptors may be of benefit in many of these diseases by reducing excessive inflammation and immune system responses. In the case of HIV infection, chemokines and antagonists which bind to HIV coreceptors may have utility in inhibiting viral entry into cells. The chemokines themselves, or agonists of their receptors, may also have utility in enhancing leukocyte movements in certain situations, such as mobilization of progenitor or stem cells out of the bone marrow into the bloodstream, where they can be harvested prior to cancer chemotherapy and then later be used to replenish cells killed during chemotherapy.
This invention encompasses the novel discovery that chemokine CK.beta.-9 uses CCR7 as its receptor. We discovered this receptor/ligand relationship by conducting experiments that show that human CK.beta.-9 polypeptide (SEQ ID NO: 2) induces calcium mobilization in human CCR7 (SEQ ID NO: 1) cell line. Therefore, this invention enables the screening of compounds that can either agonize or antagonize CK.beta.-9 and/or CCR7 and interactions thereof. We claim these screening methods and any antagonists and agonists discovered using them. We also disclose the discovery that CK.beta.-9 is a more potent ligand for CCR7 than is CK.beta.-11.
Chemokine CK.beta.-9 (also known as SLC, 6Ckine, and exodus-2) was previously known to be highly expressed in lymphoid tissues at the mRNA level, and to be a chemoattractant for T and B lymphocytes (Nagira, et al. (1997) J. Biolog. Chem. 272:19518-19524; Hromas, et al. (1997) J. Immunol. 159:2554-2558; Hedrick, et al. (1997) J. Immunol. 159:1589-1593; Gunn, etal. (1998) Proc. Natl. Acad. Sci. 95:258-263). CK.beta.-9 also induces both adhesion of lymphocytes to intercellular adhesion molecule-1 arrest of rolling cells (Campbell, et al. (1998) Science 279:381-384). All of the above properties are consistent with a role for CK.beta.-9 in regulating trafficking of lymphocytes through lymphoid tissues. CK.beta.-9 also inhibits hematopoietic progenitor colony formation (Hromas, et al., supra). Until the Applicants' discovery, no receptor for CK.beta.-9 was known. Both the amino acid and nucleic acid sequences of human CK.beta.-9 have been previously disclosed in PCT WO 96/25497, published on Aug. 22, 1996 and in PCT WO 96/06169, published on Feb. 29, 1996. CCR7 (also known as EBI1 and BLR2) (Birkenbach, etal (1993) J. Virol. 67: 2209-2220; Burgstahler, etal. (1995) Biochem. Biophys. Res. Commun. 215: 737-743) was previously known to be a receptor with only a single ligand, chemokine CK.beta.-11 (also known as ELC and MIP-3.beta.) (Yoshie, et al. (1997) J. Leukocyte Biol. 62: 634-644; Yoshida, et al. (1997) J. Biolog. Chem. 272: 13803-13809). Both the amino acid and nucleic acid sequences of human CCR7 have been previously disclosed in PCT WO 94/12519, published on Jun. 9, 1994 and in PCT WO 94/12635, published on Jun. 9, 1994. In addition, both the amino acid and nucleic acid sequences of CK.beta.-11 have been previously disclosed in U.S. Pat. No. 5,605,817.